Malaria is a major public health problem in much of the developing world. Until drug resistance developed, chloroquine was the treatment of choice for Plasmodium falciparum malaria because it was inexpensive, relatively safe and effective. Chloroquine resistance in P. falciparum results from mutations in the Plasmodium falciparum Chloroquine Resistance Transporter (PfCRT). PfCRT is an integral membrane protein localized in the parasites' digestive vacuole membrane. Sequence analysis predicts that PfCRT has ten membrane-spanning segments. PfCRT's endogenous function and its molecular interactions with chloroquine are unknown. PfCRT is an ideal anti-malarial drug target because there are no close human PfCRT homologues and because PfCRT knockout produces non-viable parasites implying that PfCRT has an essential role for normal parasite physiology. In order to screen for potential new drugs one needs a functional assay for PfCRT. A major goal of this application is to develop such a functional assay. When expressed heterologously in human embryonic kidney HEK-293 cells we showed that PfCRT is targeted to the lysosomal membrane, the digestive vacuole homologue. This application's goals are divided into two groups. The first group, Aims 1 and 2, will provide a basis for future structure-function studies of PfCRT by determining experimentally the transmembrane topology using epitope tag insertion and selective permeabilization (Aim 1) and by determining whether PfCRT is a dimer in the membrane using FRET (Aim 2). Aims 3 to'5 form the second group of experiments. They are a search for functional assays of PfCRT based on its localization in the lysosomal membrane in our heterologous expression system. These experiments are designed to identify a functional assay for PfCRT or a functional effect of PfCRT expression on lysosomal function. It is the current lack of such an assay and the fact that this is a high risk search that has motivated us to apply for an R21 grant, which is exploratory in nature, rather than an RO1.